Testosterone gel and method of use

ABSTRACT

The present invention relates to an improved transdermal hydroalcoholic testosterone gel formulation that provides, among other things, a desirable pharmacokinetic hormone profile, and methods of use.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/725,276 filed Oct. 12, 2005, the entire contents of which ishereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Testosterone, the major circulating androgen in men, is synthesized fromcholesterol. The approximately 500 million Leydig cells in the testessecrete more than 95% of the 6-7 mg of testosterone produced per day.Two hormones produced by the pituitary gland, luteinizing hormone (“LH”)and follicle stimulating hormone (“FSH”), are required for thedevelopment and maintenance of testicular function and negativelyregulate testosterone production. Circulating testosterone ismetabolized to various 17-keto steroids through two different pathways.Testosterone can be metabolized to dihydrotestosterone (“DHT”) by theenzyme 5α-reductase or to estradiol (“E2”) by an aromatase enzymecomplex.

Testosterone circulates in the blood 98% bound to protein. In men,approximately 40% of the binding is to the high-affinity sex hormonebinding globulin (“SHBG”). The remaining 60% is bound weakly to albumin.Thus, a number of measurements for testosterone are available fromclinical laboratories. The term “free” testosterone as used hereinrefers to the fraction of testosterone in the blood that is not bound toprotein. The term “total testosterone” or “testosterone” as used hereinmeans the free testosterone plus protein-bound testosterone. The term“bioavailable testosterone” as used herein refers to the non-SHBG boundtestosterone and includes testosterone weakly bound to albumin.

The following table from the UCLA-Harbor Medical Center summarizes thehormone concentrations in normal adult men range:

TABLE 1 Hormone Levels in Normal Men Hormone Normal Range Testosterone298 to 1043 ng/dL Free testosterone 3.5 to 17.9 ng/dL DHT 31 to 193ng/dL DHT/T Ratio 0.052 to 0.33 DHT + T 372 to 1349 ng/dL SHBG 10.8 to46.6 nmol/L FSH 1.0 to 6.9 mlU/mL LH 1.0 to 8.1 mlU/mL E₂ 17.1 to 46.1pg/mL

There is considerable variation in the half-life of testosteronereported in the literature, ranging from 10 to 100 minutes. Researchersdo agree, however, that circulating testosterone has a diurnal variationin normal young men. Maximum levels occur at approximately 6:00 to 8:00a.m. with levels declining throughout the day. Characteristic profileshave a maximum testosterone level of 720 ng/dL and a minimum level of430 ng/dL. The physiological significance of this diurnal cycle, if any,however, is not clear.

Male hypogonadism results from a variety of patho-physiologicalconditions in which testosterone concentration is diminished below thenormal range. The hypogonadic condition is sometimes linked with anumber of physiological changes, such as diminished interest in sex,impotence, reduced lean body mass, decreased bone density, lowered mood,and decreased energy levels.

Researchers generally classify hypogonadism into one of three types.Primary hypogonadism includes the testicular failure due to congenitalor acquired anorchia, XYY Syndrome, XX males, Noonan's Syndrome, gonadaldysgenesis, Leydig cell tumors, maldescended testes, varicocele,Sertoli-Cell-Only Syndrome, cryptorchidism, bilateral torsion, vanishingtestis syndrome, orchiectomy, Klinefelter's Syndrome, chemotherapy,toxic damage from alcohol or heavy metals, and general disease (renalfailure, liver cirrhosis, diabetes, myotonia dystrophica). Patients withprimary hypogonadism show an intact feedback mechanism in that the lowserum testosterone concentrations are associated with high FSH and LHconcentrations. However, because of testicular or other failures, thehigh LH concentrations are not effective at stimulating testosteroneproduction.

Secondary hypogonadism involves an idiopathic gonadotropin orLH-releasing hormone deficiency. This type of hypogonadism includesKallman's Syndrome, Prader-Labhart-Willi's Syndrome,Laurence-Moon-Biedl's Syndrome, pituitary insufficiency/adenomas,Pasqualini's Syndrome, hemochromatosis, hyperprolactinemia, orpituitary-hypothalamic injury from tumors, trauma, radiation, orobesity. Because patients with secondary hypogonadism do not demonstratean intact feedback pathway, the lower testosterone concentrations arenot associated with increased LH or FSH levels. Thus, these men have lowtestosterone serum levels but have gonadotropins in the normal to lowrange.

Hypogonadism may be age-related. Men experience a slow but continuousdecline in average serum testosterone after approximately age 20 to 30years. Researchers estimate that the decline is about 1-2% per year.Cross-sectional studies in men have found that the mean testosteronevalue at age 80 years is approximately 75% of that at age 30 years.Because the serum concentration of SHBG increases as men age, the fallin bioavailable and free testosterone is even greater than the fall intotal testosterone. Researchers have estimated that approximately 50% ofhealthy men between the ages of 50 and 70 have levels of bioavailabletestosterone that are below the lower normal limit Moreover, as men age,the circadian rhythm of testosterone concentration is often muted,dampened, or completely lost. The major problem with aging appears to bewithin the hypothalamic-pituitary unit. For example, researchers havefound that with aging, LH levels do not increase despite the lowtestosterone levels. Regardless of the cause, these untreatedtestosterone deficiencies in older men may lead to a variety ofphysiological changes, including sexual dysfunction, decreased libido,loss of muscle mass, decreased bone density, depressed mood, anddecreased cognitive function. The net result is geriatric hypogonadism,or what is commonly referred to as “male menopause.” Today, hypogonadismis the most common hormone deficiency in men, affecting 5 in every 1,000men. At present, it is estimated that only five percent of the estimatedfour to five million American men of all ages with hypogonadismcurrently receive testosterone replacement therapy.

SUMMARY OF THE INVENTION

The present invention relates to an improved transdermal hydroalcoholictestosterone gel formulation that provides, among other things, adesirable pharmacokinetic hormone profile, and methods of use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a standardized Pareto Chart demonstrating the effect of testfactors testosterone, isopropyl myristate and ethyl alcohol on responsevariable-viscosity.

FIG. 2 is a standardized Pareto Chart demonstrating the effect of testfactors testosterone, isopropyl myristate and ethyl alcohol on percentlabel permeated.

FIG. 3 is a standardized Pareto Chart demonstrating the effect of testfactors testosterone, isopropyl myristate and ethyl alcohol on RatioCAR.

FIG. 4 is an Estimated Response Surface Plot illustrating estimatedresponse (Ratio CAR) for a given combination of testosterone andisopropyl myristate for an alcohol (95% v/v) content of 74.3 wt %.

FIG. 5 is a Contour Plot illustrating the contours of the EstimatedResponse Surface Plot in FIG. 4.

FIG. 6 is a graph showing the cumulated amount testosterone released asa function of time for various testosterone formulations (F57 to F59) incomparison to reference formulation (F56).

FIG. 7 is a graph showing cumulative amounts of testosterone permeatedas a function of time for formulation F57.

FIG. 8 is a graph showing the cumulative amounts of testosteronepermeated as a function of time for formulation F58.

FIG. 9 is a graph showing the cumulative amounts of testosteronepermeated as a function of time for formulation F59.

FIG. 10 is a graph showing mean serum concentration-time profiles forobserved testosterone on Day 1.

FIG. 11 is a graph showing mean serum concentration-time profiles forobserved testosterone on Day 14.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in many different forms,several specific embodiments are discussed herein with the understandingthat the present disclosure is to be considered only as anexemplification of the principles of the invention, and it is notintended to limit the invention to the embodiments illustrated. Wherethe invention is illustrated herein with particular reference totestosterone, it will be understood that any other steroid in thetestosterone synthetic pathway can, if desired, be substituted in wholeor in part for testosterone in the methods, kits, combinations, andcompositions herein described.

The present invention relates to an improved testosterone gelformulation and methods of use.

In one embodiment, the present invention is directed to a method forpercutaneous administration of testosterone in a hydroalcoholic gel. Thegel comprises testosterone (or a testosterone derivative), one or morelower alcohols, such as ethanol or isopropanol; a penetration enhancingagent such as isopropyl myristate; a thickener; and water. Additionally,the present invention may optionally include salts, emollients,stabilizers, antimicrobials, fragrances, and propellants.

The present invention also includes kits, methods, combinations, andpharmaceutical compositions for treating, preventing, reversing, haltingor slowing the progression of hypogonadism or otherlow-testosterone-associated disorders in a subject once it becomesclinically evident, or treating the symptoms associated with, or relatedto the hypogonadism or low-testosterone-associated disorder. The subjectmay already have a diagnosis of hypogonadism and/or low testosterone atthe time of administration, or be at risk of developing hypogonadismand/or low testosterone. The present invention preferably is fortreatment of adult subjects over 18 years of age. Even more preferablythe present invention is for treatment of adult subjects over 21 yearsof age.

The term “derivative” refers to a compound that is produced from anothercompound of similar structure by the replacement of substitution of oneatom, molecule or group by another. For example, a hydrogen atom of acompound may be substituted by alkyl, acyl, amino, etc., to produce aderivative of that compound.

As used herein, the term “lower alcohol,” alone or in combination, meansa straight-chain or branched-chain alcohol moiety containing one toabout six carbon atoms. In one embodiment, the lower alcohol containsone to about 4 carbon atoms, and in another embodiment the lower alcoholcontains two to about 3 carbon atoms. Examples of such alcohol moietiesinclude methanol, ethanol, ethanol USP (i.e., 95% v/v), n-propanol,isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol.

As used herein, the term “ethanol” refers to C₂H₅OH. It may be used asdehydrated alcohol USP, alcohol USP, or in any common form including incombination with various amounts of water.

The composition is used in a “pharmacologically effective amount.” Thismeans that the concentration of the drug administered is such that inthe composition it results in a therapeutic level of drug delivered overthe term that the drug is to be used. Such delivery is dependent on anumber of variables including the time period for which the individualdosage unit is to be used, the flux rate of the drug from thecomposition, for example, testosterone, from the gel, surface area ofapplication site, etc. For testosterone, for example, the amount oftestosterone necessary can be experimentally determined based on theflux rate of testosterone through the gel, and through the skin whenused with and without enhancers.

In one embodiment, the present invention is directed to a method forpercutaneous administration of testosterone in a hydroalcoholic gel. Thegel comprises one or more lower alcohols, such as ethanol orisopropanol; a penetration enhancing agent; a thickener; and water. Inone embodiment, the gel comprises an anionic polymer thickening agentprecursor neutralized with a hydroxide releasing agent, such as, e.g,sodium hydroxide. Additionally, the present invention may optionallyinclude salts, emollients, stabilizers, antimicrobials, fragrances, andpropellants.

Included in the methods and pharmaceutical compositions of the presentinvention are the isomeric forms and tautomers of the describedcompounds and the pharmaceutically-acceptable salts thereof.Illustrative pharmaceutically acceptable salts are prepared from formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic,cyclohexylaminosulfonic, algenic, b-hydroxybutyric, galactaric andgalacturonic acids.

The thickening agents (aka gelling agents) suitable for use in thepresent invention include neutralized anionic polymers such aspolyacrylic acid. Preferred are the carbomer polyacrylic acids,especially those made and sold by Noveon Inc. of Cleveland, Ohio underthe trademark Carbopol®. (See information at http://www.noveon.com,incorporated herein by reference.) Particularly preferred are Carbopols®Ultrez 10, 940, 941, 954, 980, 981, ETD 2001, EZ-2 and EZ-3. Mostpreferred are Carbopol® 940 and Carbopol® 980. Other suitable anionicpolymers include carboxypolymethylene and carboxymethyl cellulose. Alsosuitable are other known polymeric thickening agents such as Pemulen®polymeric emulsifiers, and Noveon® polycarbophils. Additional thickeningagents, enhancers and adjuvants may generally be found in Remington'sThe Science and Practice of Pharmacy, Meade Publishing Co., UnitedStates Pharmacopeia/National Formulary, all incorporated herein byreference.

In one embodiment, the formulation is a gel, an ointment, a cream or apatch and is comprised of testosterone; a penetration enhancing agent,such as isopropyl myristate; a thickening agent, such as a neutralizedcarbomer; a lower alcohol, such as ethanol or isopropanol; and water.

In another embodiment, the formulation contains an anionic polymerthickening agent precursor such as a carbomer which has been combinedwith a neutralizer in an amount sufficient to form a gel in the courseof forming the composition.

In another embodiment, the formulation contains an anionic polymerthickening agent precursor such as a carbomer which has been combinedwith a neutralizer in an amount sufficient to form a gel with aviscosity greater than 9000 cps as measured by a Brookfield RV DVII+Viscometer with a spindle equal to RV6, RPM (rotations per minute) equalto 10, and the temperature maintained at 20° C.

In yet a further embodiment, the formulation contains an anionic polymerthickening agent precursor such as a carbomer which has been combinedwith a neutralizer selected from the group consisting of sodiumhydroxide, ammonium hydroxide, potassium hydroxide, arginine,aminomethyl propanol, tetrahydroxypropyl ethylenediamine,triethanolamine (“TEA”), tromethamine, PEG-15 cocamine,diisopropanolamine, and triisopropanolamine, or combinations thereof inan amount sufficient to neutralize the anionic polymer thickening agentprecursor to form a gel in the course of forming the composition.Suitable neutralizing agents and their use with selected anionic polymerthickening agent precursors are disclosed in “Neutralizing Carbopol® andPemulen® Polymers in Aqueous and Hydroalcoholic Systems,” CommercialBrochure TDS-237 (October 1998) by Noveon Inc. of Cleveland, Ohio,incorporated by reference herein.

In yet a further embodiment, the formulation contains an anionic polymerthickening agent precursor such as a carbomer which has been combinedwith a neutralizer which is an aqueous solution of sodium hydroxide suchas 0.1 N sodium hydroxide, or 1.5 N sodium hydroxide, or 2.0 N sodiumhydroxide or any other convenient strength aqueous solution in an amountsufficient to form a gel. In one embodiment, the composition wasprepared using between about 1.0% and 10.0% 0.1 N sodium hydroxide.Accordingly, embodiments employing any percentage between about 1.0% andabout 10.0% 0.1N NaOH may be used, such as, e.g., 1.0%, 2.0%, 3.0%,4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or 10.0% 0.1N NaOH.

In one embodiment the formulation is a gel and is obtained by combiningthe following substances in approximate percentages:

TABLE 2 Ingredients Combined to Yield Testosterone Formulations (% w/w)Alcohol T (95% Isopropyl Carbopol 0.1N Purified (Testosterone) v/v)Myristate 980 NaOH Water 1.20 73.5 1.00 1.0 7.00 16.3 1.40 73.5 1.00 1.07.00 16.1 1.60 73.5 1.00 1.0 7.00 15.9

In one embodiment, the composition comprises from about 1.22%testosterone to about 1.62% testosterone, such as, e.g, about 1.22%testosterone, about 1.42% testosterone, or about 1.62% testosterone.

In another embodiment, the composition comprises from about 1.15% toabout 1.22% (w/w) testosterone.

In another embodiment, the composition comprises from about 1.30% toabout 1.45% (w/w) testosterone.

In another embodiment, the composition comprises from about 1.50% toabout 1.70% (w/w) testosterone.

In one embodiment, the composition comprises about 1.15% to about 1.8%(w/w) testosterone; about 0.6% to about 1.2% (w/w) isopropyl myristate;about 60% to about 80% (w/w) alcohol selected from the group consistingof ethanol and isopropanol; a sufficient amount of a thickening agent togive the composition a viscosity in excess of about 9000 cps; and water.

In another embodiment, the composition comprises about 1.15% to about1.8% (w/w) testosterone; about 0.6% to about 1.2% (w/w) isopropylmyristate; about 67% to about 74% (w/w) alcohol selected from the groupconsisting of ethanol and isopropanol; a sufficient amount of athickening agent to give the composition a viscosity in excess of about9000 cps; and water.

The composition of the present invention can comprise about 1.15% toabout 1.25% (w/w) testosterone, about 1.30% to about 1.45% (w/w)testosterone, or about 1.50% to about 1.70% (w/w) testosterone.

In an embodiment, the viscosity of the composition of the presentinvention is about 13,000 cps to about 33,000 cps. Accordingly, theviscosity of the composition of the present invention may be any amountbetween about 13,000 cps and 33,000 cps, such as, e.g., 14,000, 15,000,16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000,25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, or33,000 cps.

In one embodiment of the present invention, the composition is obtainedby combining about 1.30% to about 1.45% (w/w) testosterone; about 0.6%to about 1.4% (w/w) isopropyl myristate; about 67% to about 74% (w/w)ethanol; about 0.6% to about 1.4% (w/w) carbomer; about 6.5% to about7.5% (w/w) 0.1N NaOH; and additional water.

In another embodiment of the present invention, the composition isobtained by combining about 1.50% to about 1.70% (w/w) testosterone;about 0.6% to about 1.4% (w/w) isopropyl myristate; about 67% to about74% (w/w) ethanol; about 0.6% to about 1.4% (w/w) carbomer; about 6.5%to about 7.5% (w/w) 0.1N NaOH; and additional water.

In yet another embodiment of the present invention, the composition isobtained by combining about 1.15% to about 1.25% (w/w) testosterone;about 0.6% to about 1.4% (w/w) isopropyl myristate; about 67% to about74% (w/w) ethanol; about 0.6% to about 1.4% (w/w) carbomer; about 6.5%to about 7.5% (w/w) 0.1N NaOH; and additional water.

The gel is rubbed or placed onto an area of skin of the subject andallowed to dry. The gel dries rapidly, i.e., within about 30 seconds toabout 3 minutes after application. Illustratively, the gel is rubbedonto an area of skin, for example, on the upper outer thigh and/or hiponce daily. Following application the subject washes his or her hands.Application of the gel results in an increased testosterone level havinga desirable pharmacokinetic profile and is effective to treat or preventhypogonadism and/or low testosterone, or the symptoms associated with,or related to hypogonadism and/or low testosterone in the subject. Thecomposition is thus useful for treating a number of conditions ordiseases.

In one embodiment, the present invention employs a packet having apolyethylene liner compatible with the components of a testosterone gel,as described below. The packet may hold a unit dose or multiple dose.

In another embodiment, the methods and compositions employ a compositionthat is dispensed from a rigid multi-dose container (for example, with ahand pump) having a larger foil packet, for example, of the compositioninside the container. Such larger packets can also comprise apolyethylene liner as above. In one embodiment, the multi-dose containercomprises an airless pump that comprises a polyethylene lined foil pouchwithin a canister with a hand pump inserted. In one embodiment, thepolyethylene lined foil pouch comprises 44 g or 88 g of product. In oneembodiment, the pump is capable of dispensing a total amount of about 75g of gel. In one embodiment, the pump is primed before use, such as,e.g., by fully depressing the pump three times and discarding the gel.In one embodiment, the pump contains enough product to allow for primingand a set number of precise doses. In one embodiment, each full pumpdepression delivers 1.25 g of testosterone gel. In this embodiment, a3.75 g dose of gel would require 3 pump depressions. A 5 g dose of gelwould require 4 pump depressions. A 7.5 g dose of gel would require 6pump depressions. A 10 g dose of gel would require 8 depressions, and soon. Of course, each pump depression can deliver any amount oftestosterone gel suitable for delivering the desired dose. The pouchsize, amount dispensed and the delivery volume per depression are notlimited to these embodiments and may be changed or adjusted to meet theneeds of the patient population.

The methods and compositions of the present invention provide enhancedtreatment options for treating, preventing, reversing, halting orslowing the progression of hypogonadism or anotherlow-testosterone-associated disorder in a subject, for example, a man,as compared to those currently available.

In one embodiment, the pharmaceutical composition of the presentinvention is administered once, twice, or three times a day, or as manytimes necessary to achieve the desired therapeutic effect. In anotherembodiment the composition of the present invention is administeredonce, twice, or three times a day on alternate days. In anotherembodiment the composition of the present invention is administeredonce, twice, or three times a day on a weekly, biweekly, or monthlybasis.

In one embodiment, a therapeutically effective dose is between about 1.0g and 10.0 g, preferably between about 1.25 g and 6.25 g.

Besides being useful for human treatment, the present invention is alsouseful for veterinary treatment of mammals, reptiles, birds, exoticanimals and farm animals, including mammals, rodents, and the like. Inone embodiment, the mammal includes a primate, for example, a human, amonkey, or a lemur, a horse, a dog, a pig, or a cat. In anotherembodiment, the rodent includes a rat, a mouse, a squirrel or a guineapig.

The composition is capable of releasing the steroid after applying thecomposition to the skin at a rate and duration that delivers in oneembodiment of the present invention at least about 10 μg per day of thesteroid to the blood serum of the subject.

In another embodiment of the present invention, the composition iscapable of releasing the testosterone after applying the composition tothe skin of a subject at a rate and duration that achieves a circulatingserum concentration of testosterone greater than about 300 ng per dlserum.

In another embodiment of the present invention, the composition iscapable of releasing the testosterone after applying the composition tothe skin of a subject at a rate and duration that achieves a circulatingserum concentration of testosterone greater than about 300 ng per dlserum during a time period beginning about 0.5 hours afteradministration and ending about 24 hours after administration.

In another embodiment of the present invention, the composition iscapable of releasing the testosterone after applying the composition tothe skin of a subject at a rate and duration that achieves a circulatingserum concentration of the testosterone between about 298 ngtestosterone per dl serum to about 1043 ng testosterone per dl serum.

In another embodiment of the present invention, after administration ofthe composition, the serum testosterone concentration is maintainedbetween about 400 and 1050 ng testosterone per dl serum.

In yet another embodiment of the present invention, after administrationof the composition, the serum testosterone concentration is maintainedbetween about 200 and 1800 ng testosterone per dl serum.

In another embodiment of the present invention, after administration ofthe composition, an obtained C_(max) is between about 300 and 5000ng/dl.

In another embodiment of the present invention, the composition isprovided to a subject for daily administration in about a 1.25 g toabout a 3.75 g dose, such as, e.g., about 1.25 g, or about 2.50 g, orabout 3.75 g. Any other suitable dose may be also be administered.

In yet another embodiment of the present invention, the subject in needof treatment has a serum testosterone level before the first application(pretreatment) of the composition of the present invention of less thanabout 300 ng/dl.

In another embodiment of the present invention, where after at leastabout 30 days of daily administration of the composition of the presentinvention the serum testosterone concentration in a subject is at leastabout 300 ng/dl to about 1050 ng/dl, such as, for example, about 300ng/dl to about 400 ng/dl, about 300 ng/dl to about 500 ng/dl, about 500ng/dl to about 700 ng/dl, about 700 ng/dl to about 900 ng/dl, about 400ng/dl to about 500 ng/dl, about 500 ng/dl to about 600 ng/dl, about 600ng/dl to about 700 ng/dl, about 700 ng/dl to about 800 ng/dl, about 800ng/dl to about 900 ng/dl, about 900 ng/dl to about 1000 ng/dl, about1000 ng/dl to about 1100 ng/dl, about 400 ng/dl to about 1050 ng/dl,about 500 ng/dl to about 1050 ng/dl, about 600 ng/dl to about 1050ng/dl, or about 700 ng/dl to about 1050 ng/dl.

In still another embodiment of the present invention, where after dailyadministration of the composition of the present invention the totaltestosterone concentration in a subject is greater than about 300 ng/dl.In one embodiment, the total serum testosterone concentration in thesubject is greater than about 400 ng/dl, about 500 ng/dl, about 600ng/dl or about 700 ng/dl. In one embodiment, the total testosteroneconcentration is measured after 24 hours of administration. In oneembodiment, the total testosterone concentration is measured after morethan 2 days of daily administration, such as, for example, after 10days, 14 days, 20 days, or 30 days.

In another embodiment of the methods, kits, combinations, andcompositions of the present invention, the composition of the presentinvention is administered once, twice, or three times daily to a subjectfor at least about 7 days. In one embodiment, the composition isadministered once a day.

Example 1 Development of Improved Testosterone Gel(s) Introduction

In order to develop a new testosterone gel formulation, a number ofexploratory studies were conducted to prepare and test gel formulationscontaining different levels of testosterone, isopropyl myristate andethyl alcohol. Preliminary studies have demonstrated that viscosity ofthe gel can be increased by slightly increasing the concentrations ofgelling and neutralizing agents. A statistical program was used togenerate a design to study the effect of 3 ingredients, testosterone,ethyl alcohol and isopropyl myristate on viscosity and in vitropermeation of testosterone from hydroalcoholic gels. In vitro permeationstudies were conducted using Franz diffusion cells. The concentration oftestosterone present in receptor samples was analyzed by HPLC techniqueor beta scintillation counter (for radiolabeled technique). Based onresults from these studies three optimized formulations were preparedand tested for skin permeation using HPLC method. All three optimizedformulations showed significant improvement in viscosity and in vitroskin permeation compared to currently marketed formulation (1%testosterone gel).

Objectives

The present disclosure summarizes studies conducted to developtestosterone gel formulation(s) with improved viscosity, reduced volumeof application, and improved in vitro skin permeation compared tocurrently marketed formulation (1% testosterone gel), and potentiallyreduce the volume of gel application.

Procedure

a. Statistical Design

A statistical design was created (StatGraphics Plus 5.1) to study theeffect of 3 ingredients, testosterone, ethyl alcohol and isopropylmyristate on viscosity and in vitro permeation of testosterone fromhydroalcoholic gels. Concentration of two other ingredients, Carbopol980 and 0.1N sodium hydroxide solution were kept constant. Following isthe design summary:

Design class: Response Surface

Design name: Box-Behnken design

Number of experimental factors: 3 (all continuous)

Number of blocks: 1; Number of runs: 15 (randomized)

Error degrees of freedom: 5

Factors Low (%) High (%) Testosterone 1.33 2.0 Isopropyl myristate 0.51.0 Alcohol (95% v/v) 72.5 76.1

The following table summarizes the ingredients of test formulations ascreated by the statistical design. These formulations were prepared at 1kg size and packaged in to glass jars for analytical and skin permeationtests.

TABLE 3 Ingredients Combined to Yield Test Formulations and ControlFormulation (% w/w) F# T Alcohol Iso- Car- Puri- (Formu- (Testos- (95%propyl bopol 0.1N fied lation) terone) v/v) Myristate 980 NaOH Water 411.665 74.3 0.75 1.0 7.00 15.3 42 1.665 72.5 0.50 1.0 7.00 17.3 43 1.66576.1 0.50 1.0 7.00 13.7 44 2.000 74.3 0.50 1.0 7.00 15.2 45 1.330 74.30.50 1.0 7.00 15.9 46 1.330 76.1 0.75 1.0 7.00 13.8 47 2.000 74.3 1.001.0 7.00 14.7 48 1.665 74.3 0.75 1.0 7.00 15.3 49 1.330 74.3 1.00 1.07.00 15.4 50 2.000 72.5 0.75 1.0 7.00 16.8 51 1.665 76.1 1.00 1.0 7.0013.2 52 2.000 76.1 0.75 1.0 7.00 13.2 53 1.665 72.5 1.00 1.0 7.00 16.854 1.330 72.5 0.75 1.0 7.00 17.4 55 1.665 74.3 0.75 1.0 7.00 15.3 56(control) 1.000 72.5 0.50 0.9 4.75 20.4

b. Analytical Testing

All test formulations and control samples were analyzed for physical(appearance, pH and viscosity) and chemical (assays for testosterone,isopropyl myristate and alcohol) attributes.

c. In Vitro Skin Permeation Studies

Permeation of testosterone was studied quantitatively with human skinplaced on the Franz diffusion cell. The skin was mounted horizontallybetween the donor and receptor half. The surface area of the skinexposed to the formulation in the donor chamber was 0.64 cm², and thereceptor volume was 5.0 mL. Temperature was maintained at 37° C. withthe help of a double water circulation jacket surrounding the lower partof the cell. The donor chamber was open on the top.

Radiolabel Method

Test formulations were spiked with ¹⁴C labeled testosterone. Spiked(radiolabeled) formulation (5-15 mg of gel containing 0.125-0.250 μCi)was applied over the surface of the epidermis gravimetrically. Periodicsamples (0, 1, 2, 4, 6, 8, 10, 22 and 24 h) were taken from the receptorcell to measure the radioactivity/amount of drug permeated across theskin. In addition the amount of radiolabel/drug remaining on the skin,in the skin samples was also determined. Further details of theseexperiments and results are presented in Example 2.

HPLC Method

Formulation (300 mg±5% which contains 3000 μg of the drug based on 1%gel) was applied over the surface of the epidermis gravimetrically.Aliquots were collected periodically (0, 1, 2, 4, 6, 8, 10, 22 and 24 h)and replaced with fresh buffer. Later aliquots were analyzed fortestosterone content. Further details of these experiments and resultsare presented in Example 3.

d. Data Analysis

In addition to data reported in corresponding examples, data from bothradiolabel and HPLC methods was analyzed further by statistical program(StatGraphics Plus 5.1). StatGraphics program was also used to predictoptimum levels of different factors which could provide maximumresponse.

Results and Discussion

Analytical Data

All test formulations were clear and have pH between 5.68-5.82. Thecontents of testosterone, isopropyl myristate and alcohol were close tothe target. The following table summarizes analytical test results after1 month storage at 40° C./75% RH.

TABLE 4 Analytical Results for Test Formulations and ControlFormulation* F# Vis- Assay- Assay- Assay- (Formu- Appear- cosity TAlcohol IPM lation) ance pH (cps) (%) (95% v/v) (%) 41 Clear gel 5.7923567 1.69 73.8 0.67 42 Clear gel 5.76 26900 1.66 71.8 0.45 43 Clear gel5.82 23000 1.69 75.5 0.44 44 Clear gel 5.75 26700 2.00 74.1 0.44 45Clear gel 5.76 25467 1.32 73.7 0.44 46 Clear gel 5.69 30233 1.40 74.50.74 47 Clear gel 5.78 24733 2.02 73.3 0.92 48 Clear gel 5.79 24767 1.6674.3 0.68 49 Clear gel 5.76 24300 1.33 73.8 0.93 50 Clear gel 5.72 261332.02 71.7 0.68 51 Clear gel 5.82 20700 1.75 75.4 0.93 52 Clear gel 5.8319733 2.00 75.5 0.68 53 Clear gel 5.72 26033 1.68 71.7 0.95 54 Clear gel5.69 28267 1.34 71.9 0.69 55 Clear gel 5.77 23233 1.67 74.0 0.68 56(control) Clear gel 5.59 22033 1.01 72.4 0.44 *test results after 1month storage at 40° C./75% RH T = testosterone, IPM = isopropylmyristate

As one of the objectives for this study is to increase the viscosity ofthe gel, statistical analysis was performed to assess the effect of testfactors testosterone, isopropyl myristate and ethyl alcohol on responsevariable-viscosity. The following is an analysis summary:

TABLE 5 ANOVA for Viscosity Analysis of Variance for Viscosity SourceSum of Squares Df Mean Square F-Ratio P-Value A:T 9.41613E6 1 9.41613E61.58 0.2488 B:IPM 3.16945E6 1 3.16945E6 0.53 0.4892 C:EtOH 1.94337E7 11.94337E7 3.27 0.1137 AA 1.43962E6 1 1.43962E6 0.24 0.6379 AB 3.37413E61 3.37413E6 0.57 0.4760 AC 3.50601E7 1 3.50601E7 5.89 0.0456 BB1.00605E6 1 1.00605E6 0.17 0.6933 BC 1.95303E6 1 1.95303E6 0.33 0.5847CC 286812.0 1 286812.0 0.05 0.8325 Total error 4.16608E7 7 5.95155E6Total (corr.) 1.17733E8 16

Referring now to FIG. 1, where in the chart A represents testosterone, Brepresents isopropyl myristate, C represents EtOH, and joint lettersrepresents a combination of factors, it is clear that the combination ofalcohol and testosterone (i.e., AC) has a significant negative effect onviscosity. This observation is consistent with earlier studies withalcohol; to maximize the viscosity the level of alcohol should be at thelowest level possible.

Permeation Data: Radiolabel Technique

Skin samples from two donors were used in this study. To minimizevariability between skins, permeation data, % label permeated (% LP),from test formulations was normalized to control formulation tested withcorresponding donor skin. Additional statistical analysis was performedon the ratio of % LP (test/control, Ratio % LP) to obtain trends andoptimal concentrations of testosterone, isopropyl myristate and ethylalcohol. Following is the analysis summary:

TABLE 6 Radiolabel Data Table used for Statistical Analysis * F# Alcohol% Ratio (Formulation) Donor T IPM (95% v/v) LP % LP 41 8127 1.665 0.7574.3 4.86 0.83 42 8127 1.665 0.50 72.5 3.45 0.59 43 8127 1.665 0.50 76.12.7 0.46 44 8127 2.000 0.50 74.3 3.8 0.65 45 8300 1.330 0.50 74.3 3.510.46 46 8300 1.330 0.75 76.1 6.37 0.84 47 8300 2.000 1.00 74.3 3.57 0.4748 8300 1.665 0.75 74.3 5.38 0.71 49 8300 1.330 1.00 74.3 9.47 1.25 508300 2.000 0.75 72.5 5.69 0.75 51 8300 1.665 1.00 76.1 6.73 0.89 52 83002.000 0.75 76.1 2.22 0.29 53 8300 1.665 1.00 72.5 4.61 0.61 54 83001.330 0.75 72.5 4.33 0.57 55 8300 1.665 0.75 74.3 4.33 0.57 56 (control)8127 1.000 0.50 72.5 5.87 1.00 56 (control) 8300 1.000 0.50 72.5 7.561.00 * % LP data from Example 2. Ratio % LP calculated from % LP values.T = testosterone, IPM = isopropyl myristate, LP = label permeated

TABLE 7 ANOVA for Ratio of % Label Permeated (Ratio % LP) Analysis ofVariance for Viscosity Source Sum of Squares Df Mean Square F-RatioP-Value A:T 0.213314 1 0.213314 7.19 0.0315 B:IPM 0.107294 1 0.1072943.62 0.0990 C:EtOH 0.00430679 1 0.00430679 0.15 0.7145 AA 0.119589 10.119589 4.03 0.0847 AB 0.131132 1 0.131132 4.42 0.0736 AC 0.0554353 10.0554353 1.87 0.2140 BB 0.00724917 1 0.00724917 0.24 0.6363 BC0.0800686 1 0.0800686 2.70 0.1445 CC 0.00941549 1 0.00941549 0.32 0.5908Total error 0.207716 7 0.0296737 Total (corr.) 0.947084 16

The Pareto chart in FIG. 2 shows that the level of testosterone hassignificant negative effect, and the level of isopropyl myristate has apositive (not statistically significant) effect on % label permeated.Above analysis suggests that the maximum level of testosterone in gelformulation should be less than highest level studied. This analysisalso suggests that the maximum level of isopropyl myristate in gelformulation should be close to the highest level studied.

Permeation Data: HPLC Technique

Same skin samples (two donors) used in radiolabel study were used inthis study. To minimize variability between skins, permeation data (fluxor cumulative amount released, CAR) from test formulations wasnormalized to control formulation tested with corresponding donor skin.Additional statistical analysis was performed on the ratio of CAR(test/control, Ratio CAR) to obtain trends and optimal concentrations oftestosterone, isopropyl myristate and ethyl alcohol. Following is theanalysis summary:

TABLE 8 HPLC Data Table used for Statistical Analysis * F# Alcohol Ratio(Formulation) Donor T IPM (95% v/v) CAR CAR 41 8127 1.665 0.75 74.3189.81 1.02 42 8127 1.665 0.50 72.5 188.21 1.01 43 8127 1.665 0.50 76.150.11 0.27 44 8127 2.000 0.50 74.3 151.95 0.82 45 8300 1.330 0.50 74.374.13 0.69 46 8300 1.330 0.75 76.1 117.87 1.09 47 8300 2.000 1.00 74.3114.61 1.06 48 8300 1.665 0.75 74.3 208.82 1.94 49 8127 1.330 1.00 74.3298.76 1.61 50 8127 2.000 0.75 72.5 94.71 0.51 51 8127 1.665 1.00 76.1254.57 1.37 52 8127 2.000 0.75 76.1 113.93 0.61 53 8127 1.665 1.00 72.5326.46 1.76 54 8127 1.330 0.75 72.5 200.45 1.08 55 8127 1.665 0.75 74.3240.61 1.30 56 (control) 8127 1.000 0.50 72.5 185.71 1.00 56 (control)8300 1.000 0.50 72.5 107.79 1.00 * T = testosterone, IPM = isopropylmyristate, CAR = cumulative amount released

TABLE 9 ANOVA for Ratio of Cumulative Amount Released (Ratio CAR)Analysis of Variance for Viscosity A:T 0.452519 1 0.452519 3.27 0.1135B:IPM 1.01713 1 1.01713 7.35 0.0302 C:EtOH 0.18431 1 0.18431 1.33 0.2864AA 0.164241 1 0.164241 1.19 0.3120 AB 0.0291287 1 0.0291287 0.21 0.6603AC 0.0600202 1 0.0600202 0.43 0.5312 BB 0.000234383 1 0.000234383 0.000.9683 BC 0.0737582 1 0.0737582 0.53 0.4891 CC 0.200542 1 0.200542 1.450.2678 Total error 0.968701 7 0.138386 Total (corr.) 3.03859 16

The results in Table 8 were subjected to a regression analysis andgenerated the following algorithm:

Ratio CAR=5.1239−0.4403*T+1.5781*IPM−0.0607*EtOH

where T is an amount of testosterone % (w/w), IPM is an amount ofisopropyl myristate % (w/w), and EtOH is an amount (w/w) of alcohol 95%v/v.

In one embodiment of the invention, values of T, IPM and EtOH areselected from within the ranges given below such that the abovealgorithm gives a Ratio CAR value greater than 1, preferably greaterthan 1.1, or most preferably greater than 2. The ranges are: between 1.0and 2.0% (w/w) testosterone, preferably between 1.15 and 1.8% (w/w)testosterone; between 0.2% and 2.0% (w/w) isopropyl myristate,preferably between 0.6 and 1.2% (w/w) isopropyl myristate; and betweenabout 60.0% and 80% (w/w) alcohol 95% v/v, preferably between about72.5% and 76.1% (w/w) alcohol 95% v/v.

Referring now to the Pareto chart in FIG. 3, statistical analysisclearly shows that the level of isopropyl myristate has significantpositive effect and the level of testosterone has negative (notstatistically significant) effect on Ratio CAR. FIG. 3 suggests that themaximum level of testosterone in gel formulation should be less thanhighest level studied. This analysis also suggests that the maximumlevel of isopropyl myristate in gel formulation should be close to thehighest level studied. Permeation results from HPLC method arequalitatively consistent with results from radiolabel method.

Response Optimization

Permeation results from HPLC method are qualitatively similar to thosefrom radiolabel method. For convenience, data from HPLC study was usedto predict (statistical optimization) optimum levels of testosterone,isopropyl myristate and alcohol for a given response. The statisticalprogram produced the following combination of factor levels whichmaximizes the ratio of cumulative amount released (Ratio CAR).

TABLE 10 Optimized Factor Levels for Cumulative Amount Released (RatioCAR) Goal: maximize Ratio CAR Optimum Value = 1.81748 Factor Low HighOptimum T 1.0 2.0 1.28995 IPM 0.5 1.0 1.0 EtOH 72.5 76.1 73.7366

FIGS. 4 and 5 illustrate estimated response (Ratio CAR) for a givencombination of testosterone and isopropyl myristate at an alcohol 95%v/v level of 74.3% (w/w).

a. Formulation Selection

Based on response surface plots and predicted optimum factor levels, thefollowing 3 formulations were selected for further permeation studies.Again for convenience these 3 formulations were tested by HPLC methodonly.

TABLE 11 Ingredients Combined to Yield Selected Formulations and ControlFormulation (% w/w) F# T Alcohol Iso- Car- Puri- (Formu- (Testos- (95%propyl bopol 0.1N fied lation) terone) v/v) Myristate 980 NaOH Water 571.20 73.5 1.00 1.0 7.00 16.3 58 1.40 73.5 1.00 1.0 7.00 16.1 59 1.6073.5 1.00 1.0 7.00 15.9 56 (control) 1.00 72.5 0.50 0.9 4.75 20.4

The following table summarizes initial (after preparation) analyticaltest results for selected formulations.

TABLE 12 Analytical Test Results for Selected Formulations and ControlFormulation F# Vis- Assay- Assay- Assay- (Formu- Appear- cosity TAlcohol IPM lation) ance pH (cps) (%) (95% v/v) (%) 57 Clear gel 5.6624500 1.21 73.1 0.94 58 Clear gel 5.71 25533 1.42 72.7 0.94 59 Clear gel5.68 24800 1.61 73.3 0.94 56 (control) Clear gel 5.57 20267 1.02 71.80.43

The following table and FIG. 6 summarize permeation data from 3 selectedformulations (data points for FIG. 6 and the table were obtained fromExample 4, Tables 17-20).

TABLE 13 HPLC Data Table used for Statistical Analysis* Time F56 (1%gel) F57 (1.2% gel) F58 (1.4% gel) F59 (1.6% gel) (hr) CAR SD CAR SD CARSD CAR SD 0 0 0 0.0 0 0.0 0.00 0.0 0 1 0 0 0.5 0.7 0.0 0.00 0.0 0 2 0 01.3 0.5 1.3 1.2 0.5 0.3 4 0.40 0.1 4.3 1.3 4.0 1.0 2.9 1.3 6 1.52 0.314.2 4.5 11.0 2.7 13.8 4.6 8 6.96 1.5 38.0 11.5 35.9 11.0 43.5 10.8 1018.35 3.4 57.0 17.9 65.4 14.3 64.5 10.4 22 48.20 12.5 119.2 51.0 129.111.8 137.5 30.6 24 50.76 12.3 129.7 50.4 137.0 13.9 156.3 36.4 *Datapoints obtained from Example 4, tables 17-20.

All 3 selected formulations showed significantly improved permeation(2-3× cumulative amount released) than control. These results furthersupport the observations from initial screening of formulations andformed the basis for selection of final formulations.

Conclusions

Statistical program was used to design the experiments based on 3 keyfactors i.e., testosterone, isopropyl myristate and ethyl alcohol. Theprogram was also used to analyze the analytical and in vitro skinpermeation data, and identify trends and optimum levels of each of thefactors to maximize response (permeation).

Three selected testosterone gel formulations have higher viscosity(−4,000 cps) than control formulation.

Significantly improved in-vitro permeation of testosterone (2-3 timesthan control) through the dermatomed human skin was observed with thethree selected testosterone gel formulations.

Example 2 In Vitro Percutaneous Absorption of Experimental TestosteroneGel Formulations through Human Skin by Radiolabel Method Materials

Formulations were prepared and supplied by Solvay Pharmaceuticals.Testosterone (¹⁴C) was procured from American Radiolabeled ChemicalsInc, (St Louis, Mo.). All other chemicals and reagents were procuredfrom approved vendors and were of highest quality and purity available.

Methods

Description of Transdermal Diffusion Cell Apparatus

The transdermal diffusion cell apparatus used in this study (PermeGear,Bethlehem, Pa.) holds up to 9 diffusion cells in series and the receptorfluid is stirred by the magnetic bead at 600 rpm. Percutaneousabsorption in vitro was studied quantitatively with human skin placed inthe Franz diffusion cell. The skin was mounted horizontally between thedonor and receptor halves of the diffusion cell. The surface area of theskin exposed to the formulation in the donor chamber was 0.64 cm², andthe receptor cell volume was 5.0 ml.

The receptor compartment was filled with phosphate buffered saline pH7.4 (PBS) and propylene glycol (1:1) and gentamicin sulphate (50 μg/ml).A double water circulation jacket (37° C.) surrounds the receptor cellin order to have the skin temperature maintained at physiologic level.The donor chamber was open towards the external environment, thusexposing the surface of the skin to the surrounding air of thelaboratory. The relative humidity (RH) of the experimental area (aroundthe diffusion cell setup) was monitored for every experiment and thiswas found to be in the range of 35 to 45% for all the experiments.

Skin Permeation Study

Human skin (thigh region) dermatomed to 0.3 mm thickness was obtainedfrom a tissue bank (US Tissue and Cell, Salt Lake City, Utah) fromcadavers. The skin was collected within 8 h of donor death and frozen in10% w/v glycerol in normal saline. The skin was stored at −80° C. untiluse. Skin from two different donors was used in the experiments. Eachexperiment was carried out with each formulation for at least 6 timesusing the skin from one donor. The skin permeation data of formulationswas compared with that of permeation of 1% marketed gel (in 6replicates) tested on the skin of same donor as test formulations andall data were normalized to the reference (marketed) formulation.

Radiolabeled Testosterone (¹⁴C, specific activity 50-60 mCi/mmol) wasused for this purpose. This is supplied by American RadiolabeledChemicals and is 99.5% pure as ascertained by HPLC.

Radioactive gels were prepared in order to apply 0.125 to 0.250 μCi in aminimum amount of the gel that spreads 0.64 cm² of the diffusion area ofFranz cell (0.64 cm²). The minimum quantity was at least 5.0 to 15.0 mg.An appropriate amount of radioactive testosterone (12.5 μCi per 125 μlof ethanol) was evaporated in a round bottom flask until the solvent iscompletely evaporated to dryness. To this flask, 500 mg of cold gelformulation was added and vortexed for 5 minutes and allowed toequilibrate over night (12 to 16 h). This gel was further vortexed for30 minutes to obtain homogenous gels. Homogeneity of the formulation'sradioactivity was determined by the counting level of 9 exactly weighted(˜5 mg) samples (standards).

The frozen skin was thawed to room temperature by keeping the skin atambient temperature for about 30 to 45 minutes. This was then rinsedwith water to remove glycerol. The skin was then put in PBS pH 7.4 andgently agitated in a shaker (100 rpm) for 20 min to remove traces ofglycerol. The washed skin was mounted on the cells approximately 30minutes before the application of the formulations. The formulation (5to 15 mg) was applied over the surface of the epidermis gravimetricallyusing a syringe (for each determination sufficient gel was dispensed tocover the test surface and the weight of the gel dispensed wasdetermined). Periodic samples were taken from the receptor cell tomeasure the amount of drug transporting across the skin (1, 2, 4, 6, 8,10, 22 and 24 h).

Washing Procedures:

At the end of the test (24 hours), the residual drug remaining at thesurface of the skin was removed by washing the surface with 200 μl ofdifferent solvents according to the following protocol:

1st wash: Cetavlon™ alcoholic (10/90 v/v)

2nd wash: water

3rd wash: Cetavlon™ alcoholic (10/90 v/v)

4th wash: water

5th wash: water.

The application area was then wiped with a cotton wool stick (Q-tip).The washings, cotton stick and the donor cell were collected in 20 ml ofethanol and allowed to extract all radioactivity in to ethanol. Theexposed area was collected by a biopsy punch. To account for lateraldiffusion, lateral portions of skin were collected and counted forradioactivity to account for Mass balance for the experiments.

The skin of the active diffusion area as well as the lateral skin wereminced into pieces with a pair of sharp point dissecting scissors(Sigma) and digested for extraction of radioactivity, with 3 ml ofSoluene 350™ (PACKARD) for overnight.

The radioactivity contained in the samples obtained as previouslydescribed, was measured in the totality or in weighed aliquots using ascintillating liquid beta counter equipped with dedicated software.

The evaluation was performed for the standards (0.5 ml/5 mlpicofluor40™), for the receptor fluid (1.0 ml/10 ml Picofluor 40™) andfor an aliquot exactly weighed of the ethanolic solution containing thewashing solvents (0.5 ml/5 ml Picofluor 40™).

For the epidermis and dermis, after digestion, 15 ml of Hionic Fluor 30™(PACKARD) were added. The background of the count is automaticallydeducted from the counting rate of each sample in counts per minute(dpm).

Data Analysis:

The results were expressed in quantities or in percentages of appliedtestosterone, found in the different compartments. Applied quantities oftestosterone were determined from the counting levels of dilutedstandards. Each result represents the mean value of 6 experimentaldeterminations and is associated with its standard error of mean.

1. The quantity of testosterone and the % of the dose absorbed in thereceptor fluid for each time were calculated as follows:

%=(Qt/Qi)×100

where Qt represents absorbed amount at time t, and Qi, applied quantityat time 0,

2. The total quantity and corresponding % of the dose absorbed as afunction of the time (cumulated values),

3. The mean flux of testosterone permeated was calculated from the slopeof the linear portion of the Q versus time plot and expressed asμCi/cm2/h

4. The quantity and % of the administered dose, which was found in theskin and in the washing solvents.

The validity of the test was checked by balancing the radioactivitywhich is found in the different samples (this summarization should becomprised, for each test, between 90% and 110% of the applied dose).

Results and Conclusion

Table 14 shows that formulations F45, F47, F52, F53, F54 and F55permeate significantly lower amount of testosterone than F56 (P<0.001).For formulations F41, 42, 43, 44, 46, 48, 50, 51 the permeated amountsappear to be lower than F56. However, the difference between theseformulations and F56 were not statistically significant (P>0.05).However F49 permeated higher than F56 but the difference between thesetwo formulations were not statistically significant (P>0.05). The massbalance data indicate variable levels of skin retention of testosterone.Furthermore, this data also demonstrates that the total mass balance isin between 90 to 110% of the initial quantity of ¹⁴C Testosteroneapplied (Table 14).

The flux of formulations F41, F42, 46, F49 and F50, albeit appearing tobe higher than F56, the differences were not statistically significant(P>0.05). The flux of the all other formulations, were althoughappearing to be lower than control, the differences among theformulations versus F56 were not statistically significant except forF45 (P>0.05).

From this study, it is clear that the rate and extent of permeation oftestosterone through human skin of all the test formulations were lower(or similar in some cases) than the reference formulation and none ofthe test formulation demonstrated significantly higher permeation thanthe reference formulation (F56).

Constraints for Flux studies: The studies conducted here were based on afinite dose kinetics where the rate limiting step is amount of gel used.Due to our using a finite dose, a non-linear permeation profile wasobtained for most of the formulations which made us difficult tocalculate the steady state flux. Nevertheless, a 2-10 h time points wereused to calculate the flux values which is an assumption of a linearprogression of flux but in actuality, the steady state was not achievedin these experiments. Hence the AUC values are a better representationto compare the formulations than flux values.

TABLE 14 Mass Balance Studies DONOR 8127 F41 F42 F43 F44 F56 Mean SDMean SD Mean SD Mean SD Mean SD % IN WASH 72.84 19.88 82.63 12.27 95.468.71 92.22 3.68 92.01 13.45 % LATERAL SK. 1.15 2.14 0.27 0.18 0.31 0.410.36 0.29 0.59 0.40 % CTRL SKIN 10.57 4.50 6.38 5.27 4.18 1.14 5.80 2.283.52 2.39 % PERMEATED 4.86 1.53 3.45 0.43 2.70 0.70 3.80 2.05 5.87 5.05% TOTAL 89.42 92.73 102.65 102.18 101.99 DONOR 8300 F45 F46 F47 F48 F56Mean SD Mean SD Mean SD Mean SD Mean SD % IN WASH 95.41 5.29 86.91 3.1687.98 13.59 79.68 7.41 86.08 3.67 % LATERAL SK. 0.24 0.14 0.42 0.17 0.140.08 0.25 0.10 0.21 0.09 % CTRL SKIN 4.09 2.05 4.67 1.84 2.29 0.84 4.882.76 4.07 1.59 % PERMEATED 3.51 1.65 6.37 3.98 3.57 1.25 5.38 3.81 7.561.78 % TOTAL 103.25 98.37 93.98 90.19 97.92 DONOR 8300 F49 F50 F51 F52F56 Mean SD Mean SD Mean SD Mean SD Mean SD % IN WASH 79.64 5.61 88.342.30 91.04 8.28 95.51 4.07 86.08 3.67 % LATERAL SK. 0.41 0.24 0.29 0.250.54 0.59 0.23 0.21 0.21 0.09 % CTRL SKIN 8.29 2.69 5.70 2.12 6.58 1.922.67 0.84 4.07 1.59 % PERMEATED 9.47 2.14 5.69 1.72 6.73 2.28 2.22 0.407.56 1.78 % TOTAL 97.82 100.01 104.89 100.64 97.92 DONOR 8300 F53 F54F55 F56 Mean SD Mean SD Mean SD Mean SD % IN WASH 85.33 4.58 87.39 4.3790.02 2.64 86.08 3.67 % LATERAL SK. 0.20 0.08 0.35 0.33 0.09 0.05 0.210.09 % CTRL SKIN 9.14 2.93 5.88 3.69 4.37 3.29 4.07 1.59 % PERMEATED4.61 0.62 4.33 0.68 4.33 0.68 7.56 1.78 % TOTAL 99.28 97.95 98.81 97.92

Example 3 In vitro Percutaneous Absorption of Experimental TestosteroneGel Formulations through Human Skin by HPLC Method Methods

Human Skin: Human frozen skin was supplied by U.S. Tissue and Cell(Cincinnati, Ohio). Skin was shipped over dry ice and once received, itwas stored at −80° C. until use. The average thickness of the dermatomedskin was 540 μm. Each experiment was carried out in replicates of six(n=6) using the same donor for any given formulation. Also, each donorwas tested for permeation of 1% marketed gel (F56) in triplicate and alldata was normalized to this measurement.

Formulations: Formulations were prepared and supplied by SolvayPharmaceuticals. Formulations were blinded except for control/marketedproduct formula (F56).

Transport Studies: Percutaneous absorption in vitro was studiedquantitatively with human skin placed on the Franz diffusion cell. Theskin was mounted horizontally between the donor and receptor half. Thesurface area of the skin exposed to the formulation in the donor chamberwas 0.64 cm², and the receptor volume was 5.0 mL. Temperature wasmaintained at 37° C. with the help of a double water circulation jacketsurrounding the lower part of the cell. This enabled the skintemperature to be maintained at physiological level. The donor chamberwas open on the top.

The receptor compartment was filled with receptor fluid consisting ofphosphate buffered saline pH 7.4 (PBS) and propylene glycol (1:1). Theskin was mounted on the cells approximately 30 minutes before theapplication of the formulations. Formulation (300 mg±5% which contains3000 μg of the drug based on 1% gel) was applied over the surface of theepidermis gravimetrically. Samples of 0.3 ml were collected periodically(0, 1, 2, 4, 6, 8, 10, 22 and 24 h) and replaced with fresh buffer.

Assay: Samples were analyzed for testosterone content using HPLC assay.The conditions/details were as follows:

Mobile phase: Acetonitrile:Water (50:50)

Column: C18, 3μ, 150 mm Phenomenex (Nucleosil)

Injection volume: 30 μl

Flow rate: 1 ml/min

UV detection: 239 nm

Results

Referring to Table 15, the results were expressed as cumulative amountsof testosterone permeated as a function of time for the differentformulations. The table shows the cumulative amount permeated relativeto control/marketed product formula using the same human skin donor. Themean flux of testosterone permeated was calculated from the slope of thelinear portion of the CAR (Cumulative amount released) versus time plotand expressed as μg/cm²/h. The results were expressed as a ratio of fluxof test formulation and Control formulation (test/control). Thecumulative amount of drug permeated through the skin per sq. cm area wasalso compared with that of marketed formulation and expressed as a ratio(test/control). Therefore, each formulation was compared to the marketedproduct formula for its cumulative permeation and flux value and theresults are compiled in Table 15. The comparison of each formulation tothe marketed product formula was assessed for statistical significanceusing ANOVA. Mean differences with p<0.05 were considered to bestatistically significant. Raw data for each formulation relative tomarketed product formula shows the Ratio Flux and Ratio CAR as well asthe statistical conclusions.

Conclusions

Permeation of testosterone through the dermatomed human skin wasobserved with all the formulations and permeation ranged from 1 to 7%with various formulations.

Referring again to Table 15, it is shown that improved permeationrelative to marketed product formulation as determined by a comparisonof cumulative amount of drug permeated after 24 hrs and/or flux atstatistically significant levels was observed for Formulations F 48, F49 and F 53.

TABLE 15 Cum Relative Amt Standard Rel Deviation Statistically betterthan Flux (CAR) ((SD/Mean)* Ratio Ratio marketed formulation?Formulation Donor (μg/cm2/h) (μg) 100) Flux CAR Ratio Flux Ratio CAR F41(n = 4) 8127 13.03 189.81 11.87 0.65 1.02 No No F 42(n = 5) 812710.944 188.21 55.52 0.54 1.01 No No F 43(n = 6) 8127 3.44 50.11 23.910.17 0.27 No No F 44(n = 6) 8127 10.57 151.95 22.05 0.53 0.82 No No F45(n = 6) 8300 5.95 74.13 29.00 0.47 0.69 No No F 46(n = 6) 8300 11.16117.87 22.60 0.88 1.09 No No F 47(n = 6) 8300 10.85 114.61 22.57 0.861.06 No No F 48(n = 6) 8300 20.53 208.82 39.5 1.62 1.94

F 49(n = 6) 8127 20.34 298.76 18.70 1.01 1.61 No

F 50(n = 6) 8127 6.78 94.71 28.76 0.34 0.51 No No F 51(n = 6) 8127 21.37254.57 29.68 1.06 1.37 No No F 52(n = 4) 8127 8.18 113.93 40.08 0.410.61 No No F 53(n = 6) 8127 24.04 326.46 28.49 1.19 1.76 No

F 54(n = 6) 8127 14.75 200.45 37.80 0.73 1.08 No No F 55(n = 4) 812716.67 240.61 27.58 0.83 1.30 No No F 56(n = 3) 8127 20.13 185.71 23.15NA NA NA NA F 56(n = 3) 8300 12.69 107.79 7.00 NA NA NA NA

Example 4 In vitro Percutaneous Absorption of Three Testosterone GelFormulations through Human Skin by HPLC Method Methods

Human Skin: Human frozen skin was supplied by U.S. Tissue and Cell(Cincinnati, Ohio). Skin was shipped over dry ice and once received; itwas stored at −80° C. until use. The average thickness of the dermatomedskin was about 700 μm. Each experiment was carried out in replicates ofsix (n=6) using the same donor for any given formulation. Permeation ofmarketed formula testosterone gel (1%, F56) was also carried out inreplicates of six (n=6) and all data was normalized to this measurement.

Formulations: Formulations were prepared and supplied by SolvayPharmaceuticals. Formulations were blinded except for control/marketedproduct formula (F56).

Transport Studies: Percutaneous absorption in vitro was studiedquantitatively with human skin placed on the Franz diffusion cell. Theskin was mounted horizontally between the donor and receptor half. Thesurface area of the skin exposed to the formulation in the donor chamberwas 0.64 cm², and the receptor volume was 5.0 mL. Temperature wasmaintained at 37° C. with the help of a double water circulation jacketsurrounding the lower part of the cell. This enabled the skintemperature to be maintained at physiological level. The donor chamberwas open on the top.

The receptor compartment was filled with receptor fluid consisting ofphosphate buffered saline pH 7.4 (PBS) and propylene glycol (1:1). Theskin was mounted on the cells approximately 30 minutes before theapplication of the formulations. Formulation (300 mg±5% which contains3000 μg of the drug based on 1% gel) was applied over the surface of theepidermis gravimetrically. Samples of 0.3 ml were collected periodically(0, 1, 2, 4, 6, 8, 10, 22 and 24 h) and replaced with fresh buffer.

Assay: Samples were analyzed for testosterone content using HPLC assay.The conditions/details were as follows:

Mobile phase: Acetonitrile:Water (50:50)

Column: C18, 3μ, 150 mm Phenomenex (Nucleosil)

Injection volume: 30 μl

Flow rate: 1 ml/min

UV detection: 239 nm

Results

Referring to FIGS. 7-9 and Tables 17-20, the results were expressed ascumulative amounts of testosterone permeated as a function of time forthe different formulations. Each table shows the cumulative amountpermeated relative to control/marketed product formula using the samehuman skin donor. The mean flux of testosterone permeated was calculatedfrom the slope of the linear portion of the CAR (Cumulative amountreleased) versus time plot and expressed as μg/cm²/h. The result wasexpressed as a ratio of flux of test formulation and Control formulation(test/control). The cumulative amount of drug permeated through the skinper sq. cm area was also compared with that of marketed formulation andexpressed as a ratio (test/control). Therefore, each formulation wascompared to the marketed product formula for its cumulative permeationand flux value and the results are compiled in Table 16. The comparisonof each formulation to the marketed product formula was assessed forstatistical significance using ANOVA. Mean differences with p<0.05 wereconsidered to be statistically significant.

Conclusions

Permeation of testosterone through the dermatomed human skin wasobserved with the three formulations and permeation was about 3% (CAR)

Improved permeation relative to marketed product formulation asdetermined by a comparison of cumulative amount of drug permeated after24 hrs and/or flux at statistically significant levels was observed forall test formulations F57, F58 and F59.

Accordingly, utilizing the teachings of the present disclosure, ahydroalcoholic gel comprising testosterone, isopropyl myristate,ethanol, water and a sufficient amount of a thickening agent to give thegel a viscosity in excess of about 9000 cps can be prepared such thatwhen is applied to human skin mounted in a Frantz cell in an amount ofabout 300 mg, after 24 hours the flux ratio is in excess of 1, orpreferably in excess of 1.5 where the flux ratio is the ratio of flux oftestosterone expressed in amount per unit area and per unit time whichpermeates the skin when the gel is so tested to the flux of testosteronewhich permeates the skin when a gel of similar viscosity comprising 1 wt% testosterone, 0.5 wt % isopropyl myristate and 72.5 wt % alcohol 95%v/v is so tested. The hydroalcoholic gel has between 1.15 and 1.8% (w/w)testosterone; between 0.6 and 1.2% (w/w) isopropyl myristate, andbetween about 72.0 and 78.0% (w/w) alcohol 95% v/v.

TABLE 16 Summary of Testosterone Skin Permeation Data at 24 hours fromTest Formulations (F57, F58 and F59) and Control (F56) Relative StandardStatistically better than Flux Cumulative Amount Deviation marketedformulation? Formulation Donor (μg/cm2/h) Rel (CAR, μg) ((SD/Mean)* 100)Ratio Flux Ratio CAR Ratio Flux Ratio CAR Control-F 56 8126 4.20 50.7626.08 — — — — (n = 6) F 57 (n = 6) 8126 10.69 129.66 41.77 2.55 2.55 YesYes F 58 (n = 6) 8126 8.70 136.98 10.88 2.07 2.70 Yes Yes F 59 (n = 6)8126 12.67 156.32 15.25 3.02 3.08 Yes Yes

TABLE 17 Testosterone Skin Permeation Data from Control Formulation(F56) Control Flux Time(hrs) (area counts) Conc(μg/ml) Conc X DilConc*0.3 Total (5 ml) Cum. (μg) Cum-Sqcm SD (mcg/cm2/hr) 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 2.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.004137.83 0.05 0.05 0.02 0.25 0.25 0.40 0.10 0.20 6.00 15522.67 0.19 0.190.06 0.96 0.97 1.52 0.30 0.56 8.00 71183.00 0.88 0.88 0.26 4.38 4.466.96 1.49 2.72 10.00 185241.67 2.28 2.28 0.68 11.41 11.74 18.35 3.375.69 22.00 484302.17 5.97 5.97 1.79 29.83 30.85 48.20 12.49 2.49 24.00481870.50 5.94 5.94 1.78 29.68 32.49 50.76 12.29 1.28

TABLE 18 Testosterone Skin Permeation Data from Test Formulation -F57 F57 Flux Time(hrs) (area counts) Conc(μg/ml) Conc X Dil Conc*0.3 Total (5ml) Cum. (μg) Cum-Sqcm SD (mcg/cm2/hr) 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 1.00 5171.20 0.06 0.06 0.02 0.31 0.31 0.48 0.73 0.482.00 13574.00 0.16 0.16 0.05 0.81 0.83 1.30 0.53 0.82 4.00 45117.33 0.540.54 0.16 2.71 2.77 4.33 1.29 1.52 6.00 147837.67 1.77 1.77 0.53 8.879.10 14.21 4.45 4.94 8.00 393201.67 4.72 4.72 1.42 23.58 24.35 38.0411.49 11.91 10.00 571442.33 6.86 6.86 2.06 34.28 36.45 56.96 17.86 9.4622.00 1201173.50 14.41 14.41 4.32 72.05 76.28 119.19 50.99 5.19 24.001240824.00 14.89 14.89 4.47 74.43 82.98 129.66 50.45 5.24

TABLE 19 Testosterone Skin Permeation Data from Test Formulation -F58 F58 Flux Time(hrs) (area counts) Conc(μg/ml) Conc X Dil Conc*0.3 Total (5ml) Cum. (μg) Cum-Sqcm SD (mcg/cm2/hr) 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.002.00 13377.80 0.16 0.16 0.05 0.80 0.80 1.25 1.23 1.25 4.00 41996.33 0.500.50 0.15 2.52 2.57 4.01 1.03 1.38 6.00 114316.00 1.37 1.37 0.41 6.867.06 11.03 2.71 3.51 8.00 372728.67 4.47 4.47 1.34 22.36 22.97 35.8910.99 12.43 10.00 665420.00 7.98 7.98 2.39 39.91 41.86 65.41 14.32 14.7622.00 1304913.50 15.65 15.65 4.70 78.27 82.62 129.09 11.80 5.31 24.001123168.83 15.72 15.72 4.72 78.62 87.67 136.98 13.89 3.95

TABLE 20 Testosterone Skin Permeation Data from Test Formulation -F59 F59 Flux Time(hrs) (area counts) Conc(μg/ml) Conc X Dil Conc*0.3 Total (5ml) Cum. (μg) Cum-Sqcm SD (mcg/cm2/hr) 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.002.00 5610.17 0.07 0.07 0.02 0.35 0.35 0.54 0.25 0.54 4.00 30049.67 0.370.37 0.11 1.86 1.88 2.94 1.34 1.20 6.00 140427.33 1.74 1.74 0.52 8.698.82 13.79 4.64 5.42 8.00 439193.83 5.44 5.44 1.63 27.18 27.83 43.4910.80 14.85 10.00 629849.83 7.80 7.80 2.34 38.98 41.26 64.47 10.42 10.4922.00 1346943.00 16.67 16.67 5.00 83.36 87.98 137.47 30.64 6.08 24.001461065.83 18.08 18.08 5.43 90.42 100.05 156.32 36.38 9.43

Example 5 The Single and Multiple Dose Pharmacokinetics of TestosteroneAfter Administration of 1.62% Hydro-Alcoholic Gel at Dose Levels of1.25, 2.50, 3.75, 5.00, and 6.25 g in Hypogonadal Males Objectives

To determine the single and multiple dose pharmacokinetics oftestosterone after administration of testosterone gel 1.62% at doses of1.25 g (20.3 mg), 2.50 g (40.5 mg), 3.75 g (60.8 mg), 5.00 g (81.0 mg),and 6.25 g (101.3 mg).

To assess the dose proportionality and accumulation of testosterone overthe dose range of 1.25 g (20.3 mg) to 6.25 g (101.1 mg) of testosteronegel 1.62%.

Methods

Formulations: Formulations were prepared and supplied by SolvayPharmaceuticals. Formulations were blinded except for control/marketedproduct formula.

Design: A single center, open-label, randomized, single and multipledose, parallel group study in hypogonadal male subjects. Subjects wererandomized to one of five treatment groups. Each group was to becomposed of 12 subjects, for a total of 60 subjects.

Subjects who consented to participate in this study and met theinclusion/exclusion criteria were randomized to one of the followingtreatment groups:

TABLE 21 Treatment Groups Treatment Group Gel Dose (g) Testosterone Dose(mg) A 1.25 20.3 B 2.50 40.5 C 3.75 60.8 D 5.00 81.0 E 6.25 101.3

Each subject received single (Day 1) and multiple (Days 2-14) doses oftestosterone gel 1.62% over a 14-day treatment period. Study drug wasapplied topically once daily in the morning. The total duration of thestudy was 17 days, not including the screening period. Subjects wereconfined to the clinic for the entire 17-day study period. The followingtable lists the ingredients combined to yield the study formulationused.

TABLE 22 Ingredients Combined to Yield Study Formulation (% w/w)Component Function % w/w Testosterone Active pharmaceutical ingredient1.62 Alcohol (95% v/v)* Absorption enhancer 73.5 Isopropyl myristateAbsorption enhancer 1.00 Carbopol 980 Thickening agent precursor 1.000.1N Sodium hydroxide Neutralizer 7.00 Purified water Solvent 15.9*Equivalent to about 68.1% of absolute alcohol in the formulation.

Subjects: Fifty-six (56) hypogonadal males.

Main Criteria for Inclusion: Male subjects 18-75 years of age,inclusive; serum total testosterone <300 ng/dL at screening as measuredby the clinical site laboratory; and subjects with a Body Mass Index(BMI) of 20-35 kg/m̂2, inclusive.

Procedures and Assessments

Dose Administration: Testosterone gel 1.62% was applied topically oncedaily in the morning on Days 1-14. The site of application was eitherthe shoulder/upper arm area or the abdomen. The study drug was appliedin 1.25 g increments until the total target dose was reached usingmaximum surface area possible.

Twenty (20) minutes prior to the targeted time of dose application,subjects showered and washed the application site with soap and water.Subjects were not allowed to remain in the shower for longer than 10minutes. The designated area for gel application was thoroughly dried.

Site personnel directly involved with the dosing procedures wore gloveswhen handling the study gel. A fresh pair of gloves was used for eachsubject. Each incremental gel dose of 1.25 g+/−0.02 g was weighed on asheet of weighing paper on a balance. Immediately after measuring theappropriate amount of gel, the weighing paper with the measured gel dosewas wiped directly onto the subject's designated site of application bythe study personnel. The subject then rubbed the product into the skinof the designated application site using his hand. This process wasrepeated until the total target dose (1.25 g to 6.25 g) was reached.

Pharmacokinetic Sampling: Whole blood samples (10 mL each) were obtainedfrom each subject for determination of total testosterone,dihydrotestosterone, and estradiol at the following time points:

-   -   Day −1: predose, 0.5, 1, 2, 4, 6, 8, 10, 12, and 16 hours        relative to the projected time of gel application on subsequent        study days;    -   Day 1: predose, 0.5, 1, 2, 4, 6, 8, 10, 12, and 16 hours        postdose;    -   Days 2-13: predose;    -   Day 14: predose, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and 24 hours        postdose.

Bioanalysis: Serum concentrations of total testosterone,dihydrotestosterone, and estradiol were determined using validatedLC-MS/MS methodology.

Criteria for Evaluation

Safety: Vital signs, ECG, physical examination, clinical laboratorydeterminations (including PSA measurement), DRE and IPSS, safetytestosterone and hematocrit measurements.

Pharmacokinetics: For this preliminary report, pharmacokineticparameters (AUC(0-24), C_(max), C_(avg), C_(min), peak to troughfluctuation, T_(min) and T_(max)) derived from both observed andbaseline adjusted serum concentrations for testosterone.

Statistical methods: Descriptive statistics (m, mean, SD, CV, median,geometric mean, minimum, maximum) and graphical representations.

TABLE 23 Subject Demographics (Mean (range)) Treatment Group N Age(years) BMI (kg/m{circumflex over ( )}2) A (1.25 g) 11 50 (27-69) 30.4(25.6-33.6) B (2.50 g) 11 50 (31-66) 31.0 (25.9-36.1) C (3.75 g) 11 52(38-65) 29.3 (21.8-35.3) D (5.00 g) 12 55 (37-68) 29.6 (22.6-34.0) E(6.25 g) 11 48 (27-68) 30.2 (27.3-32.7) All Groups 56 51 (27-69) 30.1(21.8-36.1)

TABLE 24 Ethnicity of the Subjects (N (%)) White Not White TreatmentHispanic or Hispanic Group N Latino or Latino Black Asian A 11 5 5 0 1 B11 5 6 0 0 C 11 5 5 1 0 D 12 5 7 0 0 E 11 2 9 0 0 All 56 22 (39.3%) 32(57.1%) 1 (1.8%) 1 (1.8%) Groups

Screening Testosterone Baseline Values

All subjects at screening had testosterone concentrations <300 ng/dL,confirming the hypogonadal status of all subjects prior to exposure tostudy drug. The local clinical laboratory used chemiluminescencemethodology for these evaluation. Mean screening baseline serum totaltestosterone concentrations ranged from 215 to 232 ng/dL for the fiveindividual dose groups. Table 25 provides the screening baseline mean(range) by treatment group.

TABLE 25 Mean Screening Testosterone Baseline Values Treatment MeanBaseline Group N Testosterone (ng/dL) Range A 11 215  73-286 B 11 231132-293 C 11 230  93-295 D 12 232 132-293 E 11 225 158-282 All 56 227 23-295

Testosterone Concentration-Time Data

The mean concentration-time profiles for observed testosterone on Day 1and Day 14 are provided in FIGS. 10 and 11, respectively.

Referring to FIG. 10, on Day 1, a continuous increase in testosteroneconcentrations occurred in all treatments for approximately 8 hourspostdose. Testosterone concentrations then remained consistent over theremainder of the 24-hour dosing interval. Based on the meanconcentration-time profiles, all treatments provided sufficienttestosterone exposure to increase levels above the lower limit of theeugonadal range (>300 ng/dL) after a single dose on Day 1.

Consistent testosterone levels were observed throughout the majority ofthe 24-hour concentration-time profiles after multiple dosing oftestosterone gel 1.62%. The exception to this is Treatment D, 5.00 g,where a significant peak was observed at 6 hours postdose. This increasein the mean profile was due to the results of one subject who had areported testosterone concentration of 4980 ng/dL at 6 hours postdose.

Referring now to FIG. 11, the mean profiles on Day 14 demonstratetestosterone concentrations remain above the lower limit of theeugonadal range (>300 ng/dL) over the 24-hour dose interval for all fivedoses. An increase in testosterone exposure was observed with increaseddose over the 1.25 g to 6.25 g range, with the exception of the secondprofile peak in Treatment D, 5.00 g.

Testosterone Pharmacokinetic Results

The pharmacokinetic results for observed and baseline adjustedtestosterone are provided in Table 26 below after subjects received asingle dose of 1.62% testosterone gel on Day 1.

TABLE 26 Single Dose Pharmacokinetic Parameters for Testosterone Gel1.62% on Day 1 Treatment Gel Dose Testosterone Arithmetic Mean (SD)Parameter Group (g) Dose (mg) N Observed N Baseline Adjusted AUC(0-24) A1.25 20.3 11 7376 (1465) 11 1934 (1005) (ng*h/dl) B 2.50 40.5 11 9112(3354) 9 3168- (1845) C 3.75 60.8 11 8719 (2831) 10 3330 (1875) D 5.0081 12 11933 (3188) 12 5329 (3296) E 6.25 101.3 11 11137 (3024) 11 5573(2602) C_(avg) A 1.25 20.3 11 307 (61) 11 81 (42) (ng/dl) B 2.50 40.5 11380 (140) 9 132 (77) C 3.75 60.8 11 363 (118) 10 139 (78) D 5.00 81 12497 (133) 12 222 (137) E 6.25 101.3 11 464 (265) 11 232 (108) C_(max) A1.25 20.3 11 387 (80) 11 159 (57) (ng/dL) B 2.50 40.5 11 479 (161) 9 234(102) C 3.75 60.8 11 533 (211) 10 305 (178) D 5.00 81 12 698 (231) 12424 (200) E 6.25 101.3 11 657 (215) 11 424 (210) T_(max)[a] A 1.25 20.311 12 (4-24) 11 12 (4-24) (h) B 2.50 40.5 11 12 (4-24) 9 16 (8-24) C3.75 60.8 11 16 (8-24) 10 16 (8-24) D 5.00 81 12 12 (2-24) 12 12 (6-24)E 6.25 101.3 11 12 (8-24) 11 12 (8-24) [a]median (range).

Observed mean C_(avg) on Day 1 was in the eugonadal range of 300-1000ng/dL for all dose levels. Mean AUC and C_(avg) generally increased overthe 1.25 g to 6.25 g dose range, with similar values for Treatments Band C, and Treatments D and E, respectively. Mean C_(max) increased withdose from 1.25 g to 5.00 g, then leveled off. Median T_(max) for allgroups, except 5.00 g, was 12 hours and ranged from 2 to 24 hours.

Observed C_(max) values on Day 1 for 1.25 g, 2.50 g, and 3.75 g remainedbelow the upper limit of the eugonadal range (<1000 ng/dL). In treatmentD, 5.00 g, one subject had a C_(max) value of 1070 ng/dL. In treatmentE, 6.25 g, one subject had a C_(max) value of 1020 ng/dL. All other Day1 C_(max) values were <1000 ng/dL in Treatment groups D and E.

Baseline adjusted mean AUC and C_(avg) parameter values increased withdose over all five treatment levels. Baseline adjusted mean C_(avg)indicates endogenous testosterone concentrations increased from 81 to232 ng/dL over the 1.25 g to 6.25 g dose range after single doseadministration of testosterone gel 1.62%.

The multiple dose pharmacokinetic results for observed and baselineadjusted testosterone are provided in Table 27 below for testosteronegel 1.62% on Day 14.

TABLE 27 Multiple Dose Pharmacokinetic Parameters for Testosterone Gel1.62% on Day 14 Treatment Gel Dose Testosterone Arithmetic Mean (SD)Parameter Group (g) Dose (mg) N Observed N Baseline Adjusted AUC(0-24) A1.25 20.3 11 7731 (2914) 9 3149 (2909) (ng*h/dl) B 2.50 40.5 11 9232(4146) 11 3174- (2628) C 3/75 60.8 11 11132 (2950) 11 5346 (3834) D 5.0081 8 16115 (11345) 8 9646 (12002) E 6.25 101.3 10 15250 (4123) 10 10005(4474) C_(avg) A 1.25 20.3 11 322 (121) 9 131 (121) (ng/dl) B 2.50 40.511 385 (173) 11 132 (110) C 3.75 60.8 11 464 (123) 11 223 (160) D 5.0081 8 671 (473) 8 402 (500) E 6.25 101.3 9 634 (182) 9 413 (197) C_(max)A 1.25 20.3 11 464 (158) 9 293 (170) (ng/dL) B 2.50 40.5 11 506 (195) 11266 (119) C 3.75 60.8 11 750 (221) 11 523 (241) D 5.00 81 8 1422 (1450)8 1145 (1466) E 6.25 101.3 10 1179 (520) 10 965 (527) T_(max)[a] A 1.2520.3 11 8 (0-16) 9 4 (0.5-16) (h) B 2.50 40.5 11 4 (0-16) 11 8. (0.5-24)C 3.75 60.8 11 8 (1-12) 11 1 (1-12) D 5.00 81 8 1.5 (0.5-24) 8 1.5(0.5-24) E 6.25 101.3 10 6 (0.5-24) 10 6 (0.5-24) C_(min) A 1.25 20.3 11209 (91) 9 46 (77) (ng/dl) B 2.50 40.5 11 263 (138) 11 29 (70) C 3.7560.8 11 310 (106) 11 89 (152) D 5.00 81 8 414 (184) 8 154 (203) E 6.25101.3 10 351 (43) 10 102 (74) Fluctuation A 1.25 20.3 11 81 (20) 9 271(166) B 2.50 40.5 11 66 (18) 11 262 (188) C 3.75 60.8 11 97 (43) 11 252(149) D 5.00 81 8 129 (66) 8 355 (306) E 6.25 101.3 9 117 (52) 9 201(69) [a]median (range).

Observed AUC, C_(avg), and C_(max) parameter values on Day 14 increasedacross the dose range of 1.25 g to 5.00 g, with an apparent leveling offwith the 6.25 g dose. In the 5 g dose level group, one subject (#25791)on Day 14 had C_(max) and C_(avg) values of 4980 ng/dL and 1801 ng/dL,respectively. These values were approximately 4-fold higher than theother subjects in this same treatment group. The cause of elevatedlevels in this subject is unknown. When this subject's values areremoved from the group mean presented in the table above, thepharmacokinetic parameters for the 5.00 g dose group are reduced from1422 to 914 ng/dL for C_(max) and 671 to 510 ng/dL from C_(avg),respectively. Using these revised mean values, a trend of increasedC_(avg) and C_(max) values over the entire dose range of 1.25 g to 6.25g is observed.

Observed mean C_(ram) values remained above the lower limit of theeugonadal range (>300 ng/dL) with multiple dosing at the 3.75 g, 5.00 g,and 6.25 dose levels. Observed mean C_(avg) for all dose levels rangedfrom 322 to 671 ng/dL and were in the eugonadal testosterone range of300 to 1000 ng/dL.

Observed C_(max) values on Day 14 for the 1.25 g dose level remainedbelow the upper limit of the eugonadal range (<1000 ng/dL). In the otherdose groups, a total of 12 subjects had observed C_(max) values above1000 ng/dL. At the 2.50 dose level, one subject had a C_(max) value of1010 ng/dL. At the 3.75 dose level, one subject had a C_(max) value of1070 ng/dL. At the 5.00 g dose level, four subjects had C_(max)values >1000 ng/dL ranging from 1050 to 4980 ng/dL. At the 6.25 doselevel, six subjects had C_(max) values >1000 ng/dL ranging from 1110 to2080 ng/dL. These observations are based on the bioanalytical resultsfrom the LC-MS/MS assay. These values were not identified during thepredose testosterone safety testing conducted at the clinical site.

Baseline adjusted mean C_(avg) values increased with dose across theentire dose range. Baseline adjusted mean C_(avg) indicates endogenoustestosterone concentrations increased from 131 to 413 ng/dL over the1.25 g to 6.25 g dose range after fourteen days of multiple doseadministration of testosterone gel 1.62%.

Conclusions

Based on the preliminary review of adverse event data, safetytestosterone and hematocrit laboratory measures, and application siteevaluation, testosterone gel 1.62% was safe and well tolerated at doselevels ranging from 1.25 to 6.25 g of gel (20.3 to 101.1 mg oftestosterone). After single and multiple dose administration oftestosterone gel 1.62% at dose levels ranging from 1.25 g to 6.25 g(20.3 to 101.1 mg of testosterone), mean C_(avg) values in the eugonadalrange of 300-1000 ng/dL are obtained.

At the highest dose levels of 5.00 g and 6.25 g (81.0 and 101.3 mg oftestosterone, respectively) a greater incidence of C_(max) valuesexceeding the upper limit of normal for eugonadal men was observed.Appropriate monitoring in Phase 3 clinical development is indicated.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of individual numerical values are stated as approximations asthough the values were preceded by the word “about” or “approximately.”Similarly, the numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about” or “approximately.”In this manner, variations above and below the stated ranges can be usedto achieve substantially the same results as values within the ranges.As used herein, the terms “about” and “approximately” when referring toa numerical value shall have their plain and ordinary meanings to aperson of ordinary skill in the art to which the particular subjectmatter is most closely related or the art relevant to the range orelement at issue. The amount of broadening from the strict numericalboundary depends upon many factors. For example, some of the factorswhich may be considered include the criticality of the element and/orthe effect a given amount of variation will have on the performance ofthe claimed subject matter, as well as other considerations known tothose of skill in the art. As used herein, the use of differing amountsof significant digits for different numerical values is not meant tolimit how the use of the words “about” or “approximately” will serve tobroaden a particular numerical value. Thus, as a general matter, “about”or “approximately” broaden the numerical value. Also, the disclosure ofranges is intended as a continuous range including every value betweenthe minimum and maximum values plus the broadening of the range affordedby the use of the term “about” or “approximately.” Thus, recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it there individually recitedherein.

Use of the phrase ‘the invention’ or ‘the present invention’ is notmeant to limit the claims in any manner and no conclusion should bedrawn that any description or argument associated with a particular useof the phrase ‘the invention’ or ‘the present invention’ applies to eachand every claim. The use of the phrase ‘the invention’ or ‘the presentinvention’ has been used solely for linguistic or grammaticalconvenience and not to effect a limitation of any nature on any of theclaims.

Alternative embodiments of the claimed invention are described herein,including the best mode known to the inventors for carrying out theclaimed invention. Of these, variations of the disclosed embodimentswill become apparent to those of ordinary skill in the art upon readingthe foregoing disclosure. The inventors expect skilled artisans toemploy such variations as appropriate, and the inventors intend for theclaimed invention to be practiced otherwise than as specificallydescribed herein. Accordingly, the claimed invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the claimed invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

It is to be understood that any ranges, ratios and ranges of ratios thatcan be formed by, or derived from, any of the data disclosed hereinrepresent further embodiments of the present disclosure and are includedas part of the disclosure as though they were explicitly set forth. Thisincludes ranges that can be formed that do or do not include a finiteupper and/or lower boundary. Accordingly, a person of ordinary skill inthe art most closely related to a particular range, ratio or range ofratios will appreciate that such values are unambiguously derivable fromthe data presented herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of this disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,such as, preferred, preferably) provided herein, is intended merely tofurther illustrate the content of the disclosure and does not pose alimitation on the scope of the claims. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the claimed invention.

1-44. (canceled)
 45. A hydroalcoholic gel pharmaceutical compositionconsisting essentially of: a) about 1.62% (w/w) testosterone; b) about1.0% (w/w) isopropyl myristate; c) about 73.5% (w/w) ethanol; d) about1.0% (w/w) of a neutralized thickening agent; and e) water in an amountsufficient to make the composition 100%.
 46. The hydroalcoholic gelpharmaceutical composition of claim 45 wherein the ethanol is about 95%(v/v) ethanol.
 47. The hydroalcoholic gel pharmaceutical composition ofclaim 45, wherein the neutralized thickening agent is polyacrylic acid.48. The hydroalcoholic gel pharmaceutical composition of claim 47,wherein the neutralized polyacrylic acid is obtained by combining about1.00% (w/w) of carbomer polyacrylic acid with about 7.0% (w/w) 0.1 Nsodium hydroxide.